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Chlorella vulgaris was isolated from freshwater fish ponds with pH of 8.5 at Fisheries Education and Training Agency (FETA) in Mwanza, Tanzania, using a standard plating method. Twenty grams of agar was mixed with 1 l of autoclaved Bold’s Basal Medium (BBM) in a conical flask and boiled for 20 min. After boiling, the agar solution was poured on sterilized glass petri dishes (100 × 15 mm) and allowed to cool for 2 h. Five milliliters of algal sample was transferred into media plate and spread uniformly across the surface; the plates were then placed at a light intensity of 5000 lux under room temperature (27–30 °C). After 15 days, cell colonies were observed to grow on the surface, and the best individual colonies were picked up by using a sterile syringe needle and transferred to the culture tubes containing liquid BBM and placed at the same conditions. After 14 days, when the color change was observed in the culture tube, a sample of 5 mL was taken and checked under the light microscope to see the isolated algal strain. Identification was carried out using a guide provided by (Shubert and Gärtner 2015).
Aquaculture wastewater (AWW) was collected from African catfish pond at the University of Dar es Salaam, Department of Aquatic Science and Fisheries at Kunduchi, Dar es Salaam. Immediately after collection, the AWW was sterilized by autoclaving for 15 min at 121 °C and stored in a refrigerator (4 ± 2 °C) for 2 days for sedimentation of any visible solid particles (Zhu et al. 2013). The supernatant was collected and used as the microalgae culture medium. Water quality parameters such as temperature (T), dissolved oxygen (DO), and pH were analyzed at the time of wastewater collection using a multiparameter equipment (multi-3430 WTW, Germany). Ammonium (NH4+) and phosphorus (P) were analyzed using indophenol blue and ascorbic acid method respectively, following the procedures described by (Allen 1989), whereas nitrate (NO3) was analyzed using cadmium reduction method as described by (Emteryd 1989). The calcium (Ca), sodium (Na), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) were determined using Atomic Absorption Spectrophotometer (AA240 Varian, USA).
The microalgae were cultured using AWW in the laboratory. The inoculum of C. vulgaris was pre-cultured in 1000 mL conical flasks using BBM at a light intensity of 5000 lux, temperature (28 ± 1 °C) and constantly mixed (150 rpm). The nutrient composition and costs for 1 l of BBM medium is depicted in Table 1. At the exponential growth phase, the microalgae cells were collected and cultured in five different media. The first and second media were BBM (control) and AWW, respectively. Third, fourth, and fifth media were AWW supplemented with NPK fertilizer at concentrations of 0.1, 0.5, and 1.0 g/L, respectively. All media components were sterilized by autoclaving at 121 °C for 15 min. The microalgae were batch cultured in 1000 mL Erlenmeyer flask containing 800 mL of medium and 200 mL of C. vulgaris with the initial cell density of C. vulgaris of 0.8 × 106 cells/mL in all treatments. All experiments were conducted at a controlled environment of temperature (28 ± 1 °C) maintained using air condition, illumination intensity (5000 ± 10 lux) measured using vertex VXLM-636 light meter, photoperiod (16:8 light:dark cycle) adjusted by electricity timer and continuous aeration was provided by aerators. The pH values in all treatments were measured using a pH meter (Fisher Scientific AB 15 Accumet Basic, Singapore) and maintained at the pH of 9–10 by adding 5 M sodium hydroxide (NaOH) or 3 M hydrochloric acid (HCL). The microalgae were cultured for 20 days and all treatments were carried out in triplicate.
Table 1 Nutrient composition and cost estimation for BBM growth mediumFull size table
The estimation of cultivation media costs was done by considering only the concentration and the price of each reagent used to make 1 l of the medium. Other expenses like taxation, electricity consumption, and transport cost were not considered. The prices of all reagents and commercial fertilizer used were obtained from https://www.alibaba.com and https://www.sigmaaldrich.com. The AWW was obtained for free.
Microalgal growth was monitored by measuring dry cell weight, optical density, and photosynthetic pigments (total chlorophyll and carotenoids). The optical density (as an indicator of cell density) was determined daily using a UV–Vis spectrophotometer (UV 6305, Genway, UK) at a wavelength of 688 nm. Microalgae dry weight (biomass concentration, g/L) was determined every 2 days, where a sample of 10 mL was taken from each treatment and filtered using a pre-weighed glass fiber filter (Whatman GF/F) and oven dried at 105 °C for 24 h. The biomass (dry weight) was weighed by electronic analytical balance XPE 105 (Mettler-Toledo, Switzerland). The specific growth rate, SGR (μ, day−1) of algal culture is a measure of the increase in biomass over time and it was calculated according to (Liang et al. 2013)
$$ \mathrm{SGR}\kern0.5em =\kern0.5em \ln\ \left({W}_2-{W}_1\right)/\left({T}_2-{T}_1\right) $$
(1)
Where W2 and W1 are the biomass concentrations (g/L) at T2 and T1 respectively.
The biomass productivity, PB (g/L/day) was calculated by the method of (Liang et al. 2013)
$$ \mathrm{PB}\kern0.5em =\kern0.5em \left({B}_1-{B}_0\right)/\left({T}_1-{T}_0\right) $$
(2)
Where B0 and B1 are the mean dry biomass concentration at the times T0 and T1, respectively.
The determination of total chlorophyll content in C. vulgaris cells was done using a spectrophotometric technique, as described by (Quarmby and Allen 1989). The chlorophyll content within the algal samples was extracted by dissolving well grinded 1 g of C. vulgaris biomass into 50 mL aqueous acetone (85% v/v) and stored at 4 °C for 24 h. Twenty-five milliliters aliquot of the extract was added to 50 mL of diethyl ether in a separating funnel and mixed well. The ether layer was washed with distilled water until all the chlorophyll passed into the ether layer. The water layer was then decanted, and the ether phase was transferred into a volumetric flask and anhydrous sodium sulfate (Na2SO4) was added for drying out the water. The absorbance of ether containing chlorophyll was measured at 660 and 643 nm using the UV–Vis spectrophotometer (Shimadzu) and the total chlorophyll content of the C. vulgaris was calculated using Eq. 3.
$$ \mathrm{Total}\ \mathrm{chlorophyll}\ \left(\%\right)=\frac{\ \mathrm{C}\ \left(\mathrm{mg}/\mathrm{L}\right)\times \kern0.5em \mathrm{ether}\ \mathrm{solution}\ \left(\mathrm{mL}\right)\times \mathrm{acetone}\ \mathrm{extraction}\ \left(\mathrm{mL}\right)}{10^4\times \mathrm{acetone}\ \mathrm{aliquot}\ \left(\mathrm{mL}\right)\times \mathrm{sample}\ \mathrm{weight}\ \left(\mathrm{g}\right)} $$
(3)
Where C = chlorophylls in ether solution = 7.12 × OD660 + 16.8 × OD643: whereby OD = optical density
Due to sample size requirements, the biomass composition analysis was done only for the growth media which showed good results in growth parameters analyzed. At the end of the experiment, the C. vulgaris biomass was harvested by centrifugation at 978.02×g for 10 min. The collected biomass was then dried at 100 °C to constant weight for protein, lipid, carbohydrate, minerals, and vitamin analyses.
Total lipids from microalgae biomass were extracted based on the procedure described by (Bligh and Dyer 1959). Accurately weighed 5 g of C. vulgaris biomass was mixed with chloroform, methanol and water in the proportion of 1:1:0.8 respectively and homogenized for 2 min under oxygen-free nitrogen (OFN) with cooling. The chloroform and water were then added to give a final solvent ratio of chloroform:methanol:water solvent ratio of 2:2:1.8. The mixture was filtered to remove biomass residues and transferred to a graduated cylinder where the volume of the chloroform layer was recorded. The solvent was separated into two layers (chloroform and aqueous methanol layers) using a separating funnel. An aliquot of the lower chloroform layer was pipetted and weighed using a pre-weighed, clean, and dry evaporation dish. The solvent was then oven dried at 40 °C for 30 min for lipid recovery and the remaining lipids were cooled in a desiccator and weighed. The percentage of lipid in C. vulgaris biomass was calculated using the equation below:
$$ \mathrm{Total}\ \mathrm{lipids}\ \left(\%\right)=\frac{\ \mathrm{residue}\ \mathrm{weight}\ \left(\mathrm{g}\right)\times \mathrm{volume}\ \mathrm{of}\ \mathrm{chloroform}\ \mathrm{layer}\ \left(\mathrm{mL}\right)\times {10}^2}{\mathrm{aliquot}\ \left(\mathrm{mL}\right)\times \mathrm{sample}\ \mathrm{weight}\ \left(\mathrm{g}\right)} $$
(4)
The total protein content in the C. vulgaris biomass was determined using semi-micro Kjeldahl digestion method (Emteryd 1989; Quarmby and Allen 1989). Here, 4 g of C. vulgaris biomass was digested with a strong sulfuric acid in the presence of selenium catalyst to convert nitrogen compounds into ammonium form. The ammonium concentration was then determined by using indophenol-blue colorimetric method and the nitrogen content was calculated using the Eq. (5) below. The percentage crude protein present in algal biomass was calculated by multiplying the nitrogen content by the conventional factor of 6.25.
$$ \mathrm{N}\ \left(\%\right)=\frac{\mathrm{N}{{\mathrm{H}}_4}^{+}-\mathrm{N}\kern0.5em \left(\mathrm{mg}\right)\times \mathrm{solution}\ \mathrm{volume}\ \left(\mathrm{mL}\right)}{10^4\kern0.5em \times \mathrm{aliquot}\ \left(\mathrm{mL}\right)\times \mathrm{sample}\ \mathrm{weight}\ \left(\mathrm{g}\right)} $$
(5)
The total soluble carbohydrate (CHO) was determined in the algal biomass by using Anthrone method as described by (Allen 1989). Five grams of C. vulgaris biomass was mixed with 30 mL of distilled water in 100 mL conical flask and boiled at boiling point of water for 2 h. The sample was allowed to cool at room temperature and filtered through a Whatman filter paper No. 44. An aliquot of a clear sample solution was placed into the test tube and the anthrone reagent was added. The solution was allowed to cool, and its absorbance was measured at 625 nm. The percentage soluble carbohydrate in the C. vulgaris biomass was calculated based on the formula below.
$$ \mathrm{Soluble}\ \mathrm{carbohydrate}\ \left(\%\right)=\frac{\mathrm{C}\ \left(\mathrm{mg}\right)\times \mathrm{extraction}\ \mathrm{volume}\ \left(\mathrm{mL}\right)}{10\times \mathrm{aliquot}\ \left(\mathrm{mL}\right)\times \mathrm{sample}\ \mathrm{weight}\ \left(\mathrm{g}\right)} $$
(6)
Whereby C = mg glucose obtained from the graph
Extraction of vitamins from C. vulgaris biomass was done by mixing 0.5 g of sample with 100 mL of 95% ethanol in a conical flask. The mixture was vigorously shaken for 15 min to ensure complete extraction. The extract was centrifuged for 10 min and filtered using Whatman No. 1 filter paper. To remove ethanol and obtain clear extracts, the sample was placed in a rotary evaporator (Gmbh & Co.KG, Germany) under reduced pressure at 40 °C. The obtained extract was then kept at 4 °C until analyses. Vitamin A (as beta- carotene) was determined according to the method of (Nagata and Yamashita 1992). One hundred milligrams of dried extract was vigorously stirred with 10 mL of acetone-hexane mixture (4:6) for 1 min and filtered through Whatman No. 4 filter paper. The absorbance of the filtrate was measured at 453, 505, and 663 nm. Content of beta carotene was calculated according to the following equation:
$$ \mathrm{Beta}-\mathrm{carotene}\ \left(\mathrm{mg}/100\ \mathrm{mg}\right)=0.216\ {\mathrm{A}}_{663}-0.30{4\mathrm{A}}_{505}+0.452\ {\mathrm{A}}_{453} $$
(7)
Determination of vitamin B complex present in microalgae extracts was done according to the method of (Rajput et al. 2011). The working standard solution for the standard vitamins and microalgae samples were prepared by dissolving a known weight of the standard vitamin and extracts in a known volume of distilled water into a conical flask. Vitamins B1, B2, B3, B6, B12, and C were determined from riboflavin, nicotinamide, pyridoxine hydrochloride, cyanocabalamin, and ascorbic acid stock solutions, respectively. The absorbance of the working solutions, C. vulgaris samples, and blank were read at 430 nm for vitamin B1, 444 nm for vitamin B2, 450 nm for vitamin B3, 650 nm for vitamin B6, 530 nm for vitamin B12, and 450 for vitamin C using a UV-visible spectrophotometer (Jenway 6305).
To determine the mineral composition of C. vulgaris biomass, the biomass was digested using nitric perchloric acid method as described by (Jones 1984). Approximately 0.5 g (dry weight) of C. vulgaris biomass from each treatment was weighed into a beaker. To the samples, a mixture of 5 mL of concentrated nitric acid (HNO3) and 1 mL of per-chloric acid (HCIO4) in the ratio of 5:1 was added. The solution was heated at 120 ̊ C until the disappearance of the brown fumes which indicated the complete digestion of the organic matter. The solution was then cooled and diluted with distilled water up to 100 mL solution. The Ca, Mg, Fe, K, Mn, Na, and Zn concentration of the digested biomass were determined using Atomic Absorption Spectrophotometer (AA240 Varian, USA).
The data were presented as mean ± standard error (SE). Statistical analysis was carried out by using R software (Version 3.6.3). Normally and not normally distributed data were tested using one-way analysis of variance (ANOVA) and Kruskal-Wallis respectively. Tukey’s (ANOVA) and Dunn (Kruskal-Wallis) post hoc test were used to check the significant difference among the treatment means. A p value of less than 0.05 was considered statistically significant.
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